BINDING OF THE 60-KDA RO AUTOANTIGEN TO Y-RNA - EVIDENCE FOR RECOGNITION IN THE MAJOR GROOVE OF A CONSERVED HELIX

The 60-kDa Ro autoantigen is normally complexed with small cytoplasmic RNAs known as Y RNAs. In Xenopus oocytes, the Ro protein is also comp lexed with a large class of variant 5S rRNA precursors that are folded incorrectly. Using purified baculovirus-expressed protein, we show th at the 60-kDa Ro protein binds directly to both Y RNAs and misfolded 5 S rRNA precursors. To understand how the protein recognizes these two distinct classes of RNAs, we investigated the features of Y RNA sequen ce and structure that are necessary for protein recognition. We identi fied a truncated Y RNA that is stably bound by the 60-kDa Ro protein. Within this 39-nt RNA is a conserved helix that is proposed to be the binding site for the Ro protein. Mutagenesis of this minimal Y RNA rev ealed that binding by the 60-kDa Ro protein requires specific base pai rs within the conserved helix, a singly bulged nucleotide that disrupt s the helix, and a three-nucleotide bulge on the opposing strand. Chem ical probing experiments using diethyl pyrocarbonate demonstrated that , in the presence of the two bulges, the major groove of the conserved helix is accessible to protein side chains. These data are consistent with a model in which the Ro protein recognizes specific base pairs i n the conserved helix by binding in the major groove of the RNA. Furth ermore, experiments in which dimethyl sulfate was used to probe a nake d and protein-bound Y RNA revealed that a structural alteration occurs in the RNA upon Ro protein binding.